Label-Free Protease Detection via Enzyme-Instructed Self-Assembly
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Solution Overview
Problem
Existing assays for detecting protease activity, such as quenched probes, suffer from high background signal due to incomplete quenching, leading to low signal enhancement after protease cleavage. Additionally, these assays often require labeling of protease substrates with fluorophores, complicating synthesis and increasing costs.
Innovation Solution
The use of enzyme-instructed self-assembly (EISA) methods, specifically self-assembling polypeptides with a β-strand motif that forms an anti-parallel beta-sheet structure upon protease cleavage, allowing for label-free detection of protease activity. This involves administering self-assembling polypeptides and a β-sheet intercalating dye into an aqueous milieu, where the fluorescent signal is detected to indicate protease presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If quenched probes are used to detect protease activity, then detection capability is achieved, but background signal is high due to incomplete quenching
Solution Approach 1:
The invention extracts the fluorophore from the substrate structure and uses it as a separate self-assembling motif. The fluorophore self-assembles into aggregates that are inherently quenched, and only upon protease cleavage does the fluorophore become free to emit, thereby eliminating background signal while maintaining detection capability
Solution Approach 2:
The invention changes the physical state of the fluorophore from monomeric (emissive) to aggregated (quenched) through self-assembly. This parameter change allows the system to transition from a high background state to a low background state upon protease activation, resolving the contradiction between detection capability and background signal
2Measurement precision
If fluorophore labeling is used in protease assays, then detection sensitivity is improved, but synthesis complexity and cost increase
Solution Approach 1:
The fluorophore performs dual functions: it serves as both the detection reporter and the self-assembly motif that drives substrate organization. This self-service capability eliminates the need for separate labeling steps and complex probe synthesis, while maintaining high detection sensitivity through fluorescence
Solution Approach 2:
The fluorophore is designed to be multi-functional, serving as both the signaling molecule and the structural organizing element that directs substrate self-assembly. This universality simplifies the overall system by eliminating separate labeling components and reducing synthesis complexity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach reduces background signal, increases sensitivity, simplifies probe synthesis, and lowers costs by enabling label-free detection of protease activity through enzyme-instructed self-assembly of polypeptides.
Implementation Method 1
a β-sheet intercalating dye configured to emit a fluorescent signal is administered into the aqueous milieu and forms a complex with one or more anti-parallel β-sheet structures formed by the self-assembly of β-strand motifs. The fluorescent signal is then detected to thereby indicate the presence of the protease in the aqueous milieu.
Implementation Method 2
a self-assembling polypeptide comprises a β-strand motif configured to self-assemble with one or more nominally identical β-strand motifs and form an anti-parallel beta-sheet structure
Data Source
AI summary
The present disclosure provides self-assembling polypeptides and methods for detecting protease activity by enzyme-instructed beta-sheet formation. A self-assembling polypeptide comprises a β-strand motif configured to self-assemble with one or more nominally identical β-strand motifs and form an anti-parallel beta-sheet structure. The β-strand motif being operatively connected to a hydrophilic motif by a protease substrate motif that comprises a protease cleavage site configured to specifically hybridize with a protease. Whereby, when in an aqueous milieu and upon hybridization of the protease to the protease cleavage site, the protease cleaves the self-assembling polypeptide and dissociates the β-strand motif allowing the dissociated β-strand motif to self-assemble with the one or more nominally identical β-strand motifs and thereby form the anti-parallel β-sheet structure. A β-sheet intercalating dye is complexed with the anti-parallel β-sheet structure and detection of fluorescent signal indicates proteolytic activity.